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Bioinformatics Analysis of DNMT1 Expression and Its Role in Head And www.nature.com/scientificreports OPEN Bioinformatics analysis of DNMT1 expression and its role in head and neck squamous cell carcinoma prognosis Jili Cui1,2,4, Lian Zheng2,3,4, Yuanyuan Zhang1,2* & Miaomiao Xue1,2* Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of malignancy in the world. DNA cytosine-5-methyltransferase 1 (DNMT1) play key roles in carcinogenesis and regulation of the immune micro-environment, but the gene expression and the role of DNMT1 in HNSCC is unknown. In this study, we utilized online tools and databases for pan-cancer and HNSCC analysis of DNMT1 expression and its association with clinical cancer characteristics. We also identifed genes that positively and negatively correlated with DNMT1 expression and identifed eight hub genes based on protein–protein interaction (PPI) network analysis. Enrichment analyses were performed to explore the biological functions related with of DNMT1. The Tumor Immune Estimation Resource (TIMER) database was performed to explore the relationship between DNMT1 expression and immune-cell infltration. We demonstrated that DNMT1 gene expression was upregulated in HNSCC and associated with poor prognosis. Based on analysis of the eight hub genes, we determined that DNMT1 may be involved in cell cycle, proliferation and metabolic related pathways. We also found that signifcant diference of B cells infltration based on TP 53 mutation. These fndings suggest that DNMT1 related epigenetic alterations have close relationship with HNSCC progression, and DNMT1 could be a novel diagnostic biomarker and a promising therapeutic target for HNSCC. Head and neck squamous cell carcinoma (HNSCC), an aggressive malignancy that is difcult to diagnose early, accounts for more than 95% of head and neck cancers1,2. Most HNSCC patients are diagnosed at an advanced stage with an extremely poor prognosis. It is estimated that 400,000 new patients are diagnosed annu- ally worldwide3. Surgical resection, along with radiotherapy and chemotherapy, is recommended for early stage HNSCC. Although a comprehensive and multidisciplinary approach, including chemotherapy, radiotherapy, and immunotherapy, are used for advanced-stage HNSCC, the 5-year overall survival (OS) rate has remained below 50%4,5. Tus, by delineating the mechanisms of tumorigenesis and immune infltration in HNSCC, a foundation for future therapeutic developments will be laid. 5-methylcytosine (m5C) modifcation is an important post-transcriptional methylation, and m5C is associ- ated with the carcinogenesis and tumorigenesis of various cancers6. 5-methylcytosine regulators function as one kinds of important epigenetic modifers6 . DNA methylation plays a role in epigenetic gene silencing, X chromosome inactivation, and genome stability 7. It has also been shown that DNA methylation modifcations are crucial for normal cell cycle regulation, development, and diferentiation8. Recent studies have demonstrated that epigenetic DNA methylation alterations drive formation of many types of cancer9. DNA methylation is catalyzed by DNA methyltransferase enzymes (DNMTs), which regulate epigenetic modifcations in mammals. DNMTs methylate CpG islands in promoters, aberrant modifcation of DNMTs methylate could cause carcinogenesis and tumor progression 10. DNA cytosine-5-methyltransferase 1 (DNMT1) acts as a crucial epigenetic modifer by regulating DNA methylation of target genes, which involves in tumor suppressor gene expression11. DNMT1 gene expression is also subject to epigenetic regulation. Numerous studies have shown that DNMTI is highly upregulated in various cancers such as cholangiocarcinoma 12, hematological malignancies 13, pancreatic cancer 14, breast cancer15. DNMTI is required for maintaining the cancer stem cell status16,17. In lung cancer patients, 1Department of General Dentistry, The First Afliated Hospital, Zhengzhou University, No. 1 Jianshe Road, Zhengzhou 450052, Henan, China. 2Key Laboratory of Clinical Medicine, The First Afliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan, China. 3Department of Oral and Maxillofacial Surgery, The First Afliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan, China. 4These authors contributed equally: Jili Cui and Lian Zheng. *email: [email protected]; [email protected] Scientifc Reports | (2021) 11:2267 | https://doi.org/10.1038/s41598-021-81971-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. High DNMT1 expression was observed in head and neck cancers and in pan-cancers (A) High DNMT1 expression was found in several types of cancers. (B) Te DNMT1 transcript level was signifcantly higher in HNSCC tissue than in non-tumor tissue. (C) DNMT1 protein expression was higher in HNSCC tumor tissue than in non-tumor tissue. high levels of DNMT1 expression are associated with poor clinical prognosis 18.However, it is also reported that upregulation of DNMT1 also functions in tumor-suppressor gene activation 19,20. Further, DNMT1 regulates the immune system and is indispensable for the inhibitory function of Foxp3 + Treg cells21. In our previous study, we utilized the bioinformation analysis and validated the characters of m5C related regulators in HNSCC, and DNMT1 functioned as a key regulator in HNSCC progression 22. We identifed that DNMT1 might serve as a crucial m5C regulator in malignant activities of HNSCC, and increased DNMT1 expression was associated with patient’s mortality rate22. Nevertheless, the comprehensively biological activities of DNMT1 in HNSCC tumorigenesis and progression has not yet been clarifed. In this study, we used public databases and online analysis websites to unravel the role of DNMT1 in HNSCC tumorigenesis and progression. Furthermore we identifed methylation markers with diagnostic potential, and identifed potential targets for the treatment of HNSCC. Results DNMT1 gene expression and mutation distribution. We performed a pan-cancer analysis of DNMT1 expression, and we found that DNMT1 was upregulated in several diferent tumor types (Fig. 1A). Specifcally, transcription level of DNMT1 was signifcantly higher in HNSCC tumor tissues (n = 520) than in normal tissues (n = 44, p < 0.001) (Fig. 1B). To further validate the expression of DNMT1, the Immunohistochemistry (IHC) was applied to detect the protein expression of DNMT1 gene in normal tissues and tumor tissues based on HPA. Results showed that DNMT1 protein expression was signifcantly overexpressed in HNSCC tissues compared with normal tissue (Fig. 1C). Tus, DNMT1 had potential as a biomarker for diagnosis of HNSCC. The clinical features and prognosis of DNMT1 in HNSCC. To explore the DNMT1 related clinical characters, we determined whether gender, age, race, tumor stage, tumor grade, nodal metastasis, and human papillomavirus (HPV) infection status correlated with DNMT1 expression in HNSCC. We found that DNMT1 expression level was signifcantly higher in males patients than in females patients (p < 0.001) (Fig. 2A) and in the less than 60-year-old subgroups (n = 256), DNMT1 expression level was remarkably higher compared with over61-year-old subgroups (n = 261, p = 0.003) (Fig. 2B). We also observed that DNMT1 expression was higher in Caucasians compared with Asians and African-American subgroups (p = 0.037) (Fig. 2C). Advanced-grade tumors had higher DNMT1 gene expression level compared with lower grade tumor samples (Fig. 2D), but Scientifc Reports | (2021) 11:2267 | https://doi.org/10.1038/s41598-021-81971-5 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Relationships between DNMT1 expression and clinical characteristics and prognosis were analyzed. (A–G) DNMT1 expression correlated with gender, age, race, tumor grade, and HPV infectious status, but not with lymphatic invasion or tumor stage. (H, I) OS and RFS analyses demonstrated that patients with high DNMT1 expression had poor clinical prognosis. TNM stages and lymphatic invasion did not correlate with DNMT1 expression (Fig. 2E,F). We validated that HPV-positive patients (n = 41) had higher DNMT1 expression compared with HPV negative patients (n = 80) (p < 0.001) (Fig. 2G). Furthermore, we explored the overall survival (OS) and relapse-free survival (RFS) of HNSCC patients (Fig. 2H,I) and found that patients with high DNMT1 expression had better OS rate (HR = 0.69, logrank p = 0.0076). Patients with high expression level of DNMT1 showed no signifcant diference in RFS com- pared with low DNMT1 expression subgroups (HR = 2.4, logrank p = 0.094). DNMT1 may be potential genes for HNSCC poor prognosis prediction and may provide reference value for HNSCC treatment in the further. DNMT1 gene mutations and co-expressed genes. Te somatic mutation rate of DNMT1 in HNSCC patients was 1.6%, and the eight mutations identifed were all missense mutations, P1330S, P1325S, E912Q, S1352G, P692S, H370Y, T616M, and R325L (Fig. 3A). We also identifed eight co-related genes such as CLSPN, UHRF1, BRCA1, ATAD5, TIMELESS, CIT, KIF4B and DTL (Table 1). Te correlation between DNMT1 mutation status and the 8 genes mutation status were evaluated. In this study, the 8 genes showed signifcantly strong correlation with DNMT1 expression, as illustrated in Fig. 3B. CLSPN (Spearman: 0.78, p = 3.46e−102), UHRF1 (Spearman: 0.75, p = 2.11e−89), BRCA1(Spearman: 0.74, p = 6.34e−86), ATAD5 (Spearman: 0.73, p = 4.92e−84), TIMELESS (Spearman: 0.73, p = 3.52e−82), CIT (Spear- man: 0.71, p = 2.51e−76), KIF4B (Spearman: 0.71, p = 2.93e−75), and DTL (Spearman: 0.70, p = 4.90e−74). DNMT1 expression associates with immune cell
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